Evaluation of Virulence Factors and Detection of vanA , vanB and esp Genes from Clinical Isolates of Vancomycin - Resistant Enterococcus faecalis

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The Enterococci are a diverse and versatile group of bacteria with several intrinsic characteristics that allow them to survive and grow under a variety of conditions (Tellis and Muralidharan, 2012).Among the Enterococcal species described Enterococcus faecalis represented one of the most important causes of nosocomial infections, since it is responsible for 80 -90% of human Enterococcal infections (Jett and Gilmore, 1990; Jones et al., 2004;Fernandes & Dhanashree, 2013).
The general interest for Enterococci and treatment of Enterococcal infections has increased due to the appearance of antibiotic multi resistant strains (Dahle´n et al., 2012).One of the major reasons why these organisms have survived in the hospital environment is their intrinsic resistance to several commonly used antibiotics and, perhaps more important, their ability to acquire resistance to all currently available antibiotics, either by mutation or by receipt of foreign genetic material through the transfer of plasmids and transposons.Most Enterococci are tolerant to the bactericidal activity of antibiotics; bactericidal synergy between antibiotics is needed to treat most serious Enterococcal infections such as endocarditis and meningitis (Cetinkaya et al., 2000).There are at least three major reasons for the emergence of multidrug-resistant Enterococci: (i) baseline intrinsic resistance to several antimicrobial agents, (ii) acquired resistance via mobility of the resistance genes on plasmids and transposons, and chromosomal exchange, and (iii) the transferability of resistance (Mundy et al., 2000).Resistance to last-line drugs, such as vancomycin is common, and Enterococci are now disseminating this resistance to methicillin-resistant Staphylococcus aureus (MRSA) (Palmer and Gilmore, 2010).Among vancomycin-resistance phenotypes in Enterococci, VanA and VanB possess highest clinical importance (Sharifi et al., 2012).Glycopeptide resistance in Enterococci is mediated predominantly by mobile gene clusters that confer resistance to vancomycin and teicoplanin (Howden et al., 2013).

MATERIALS AND METHODS
One hundred thirty -five clinical specimens were collected from urine, blood, teeth root canal and burns of patients' suffering from urinary tract infection, bacteremia, endodontic infections and burns infections, respectively [Table 1].
The collected specimens were streaked on Pfizer Selective Enterococcus; and incubated at 37°C for 24 hr.VITEK system was employed for E. faecalis isolates confirmation.

Determination of minimum inhibitory concentrations [MICs] of Vancomycin
The MICs were determined to all E. faecalis isolates for the Vancomycin antibiotic.This test was achieved according to Morello et al. [12].
The MIC values were based on break point recommended by CLSI [13], for the estimation of response.For Vancomycin, 1-4µg was sensitive, more than 4µg isolate was considered as intermediate resistant and e" 32µg considered as resistant.

D e t e c t i o n o f s o m e Vi r u l e n c e f a c t o r s phenotypically
Haemolysis activity was determined by streaking of pure isolates on blood agar plates, The Egg-Yolk agar and Skim milk agar were inoculated with the E. faecalis isolates for detection lipase and protease production; respectively.Gelatin liquefaction (Harley & Prescott, 2002) activity was determined by streaking of pure isolates on gelatin nutrient medium.Aggregation substance production (Creti et al., 2004) (2) were selected for this study; these primers were provided in lyophilized form.
The extracted DNA, primers and PCR premix, were thawed at 4ºC, PCR mixture was set up in a total volume of 25 µl included: 5 µl PCR premix, 2.5 µl of each primer (forward and reverse), 5 µl of DNA template and the rest volume was completed with sterile deionized distilled water, the negative control contained all material except template DNA, so instead that deionized distilled was added.The PCR reaction tubes were mixed briefly and placed into thermocycler PCR instrument where DNA was amplified according to the PCR program as indicating in Tables 3, 4, 5.
Determination of PCR Specificity The PCR products were analyzed and the result confirmed by using 1.5 % agarose gel electrophoresis, by dissolving 1.5 gm.

Isolation and Identification of Vancomycin-Resistant E. faecalis
Twenty isolates of the genus Enterococcus were isolated from 135 clinical specimens have the ability to grow on Pfizer selective Enterococcus.The highest numbers of isolates were distributed among urine specimens and the lowest one was observed among wound infection specimens, VITEK were employed to confirm the presence of E. faecalis isolates, regarding samples' type and isolated Enterococcus spp., there were a 20 isolates identified, they were demonstrated in table 7.
E. faecalis isolates from urine in this study was 46.6%, Alebouyeh et al. [19] showed that the percentage of E. faecalis isolates from urine was 75%.
For blood specimens, the percentage of E. faecalis isolates were 8.3%, this result was compatible with the results by Tellis and Muralidharan [2]; they showed that percentage of E. faecalis isolated from blood was 18%.While other studies reported by Al-Jarousha et al. [21], AL-khafaji et al. [22] and Mira et al. [23] revealed that the percentage of E. faecalis isolated from blood were 3%, 0% and 55.05%, respectively.
The percentage of E. faecalis isolated from root canal specimens was 14%, this result was higher than the result of local study obtained by Mahmoudpour et al. [24], who showed the percentage of E. faecalis isolated from root canal was 10%, another studiy by Zoletti et al. [25] and Preethee et al. [26], showed that the percentage of E. faecalis isolated from root canal was 80% and 46.87%, respectively.The percentage of E. faecalis isolated from wounds was 10%, this result was compatible with the result of Giacometti et al. [27] and Al-Jarousha et al. [21], who indicated that the percentage of E. faecalis isolated from wounds was 5.6%, 1.9%, respectively.
The differences between isolation percentages may be related to the number of specimens, the differences in the source of isolates, hospitals included in each study, their geographical regions and differences in the identification methods.

Determination of Vancomycin susceptibility
Vancomycin susceptibility was determined by the minimum inhibitory concentration [MIC]  In a study reported by Fatholahzadeh et al. [28] stated that 38% of E. facalis isolates were resistant to vancomycin.While Camargo et al. [29] demostrated that 20.8% of E. faecalis isolates were resistant to vancomycin and 79.1% of isolates were sensitive.The results of this study was close to the results of Chabuck et al. [30], Al-jmor [20] and Praharaj et al. [31], who showed that the percentages of vancomycin resistant were 71.43%, 50% and 90.6%, respectively.
The multidrug resistance (MDR) bacteria was defined as resistance to 3 or more types of antibiotics (Al-Jarousha et al. 2008), the emergence of multidrug-resistant (MDR) pathogens seriously threatens this classes of lifesaving drugs (Queenan and Bush, 2007), therefore; This study was showed the percentage of multidrug resistant E. faecalis isolates was 90% as shown in table (9).This result was adjacent to the results reported by Al-Jarousha et al. (2008) and Chabuck et al. (2011) were showed the percentage of MDR is 66.6% and 100%, respectively.
Resistance to multiple classes of antibiotics is common in Enterococci as had seen in this study, this finding was alarming as infection due to MDR Enterococcal isolates are difficult to treat.Enterococci showed a great resistance to most of the commonly used antibiotics in hospitals where development of antibiotic resistance is often related to the overuse, and misuse of the antibiotics prescribed (Saifi et al., 2008).
Iraq is one of the developing countries where all types of antibiotics are sold over the counter, an attitude that encourages selfmedication.
From this study results, it can be conclude that the resistance to multiple classes of antibiotics is common to E. faecalis, these findings were alarming the infections due the MDR and extended -resistant E. facalis isolates and difficult to treat (Al-Jmor , 2012).

Detection the Virulence factors of E. faecalis
Hemolysin production was detecting by culturing the isolates on blood agar base supplemented with 5% blood, and hemolytic activity was observed as clear zone (²-hemolysis) surrounding the bacterial colonies.All the bacterial isolates showed ²-hemolysis (Figure 1).This result was similar to the result reported by Al-Jmor (2012) that showed 100% of E. faecalis isolates have the ability to produce hemolysin.Jankoska et al. (2008) reported that 50% of isolates produce hemolysin.In local studies reported by AL-Khafaji et al. (2010) and Mousa (2012) that showed the percentage of hemolysin production by E. faecalis isolates was 15.1%, 40%.Tellis and Muralidharan (2012) showed the percentage of E. faecalis isolates which have ability to produce hemolysin is 44%.
Lipase production was detected on eggyolk agar plates, the hydrolyzed clear zone around the colonies were considered a positive result.The results of this study showed that 25% of the E. faecalis isolates were lipase producer (Figure 1).AL-Khafaji et al. (2010) showed that the percentage of lipase producer was 3%; while Al-Jmor (2012) has reported 2.94% of isolates were lipase producer, whereas; the result of this study was similar to the findings of Elsner et al.The biological role of lipase in infection might be considered the most important step in bacterial infection, the most prominent role of microorganism extracellular lipases may be the digestion of host cellular lipids for nutrient acquisition, which results in sticking to host tissue and neighboring cell (Furumura et al., 2006).
Protease production was detected on skim milk agar plates; the hydrolyzed clear zones around the colonies were indicating a positive result.The results showed 100% of isolates were protease producers (Figure 1).This result was close to Furumura et al. (2006) who demonstrated 75% of E. faecalis isolates was protease producer.Al-Jmor (2012) was reported a result similar to this study, showing that 100% of E. faecalis isolates was protease producing.
Gelatinase is extracellular metalloprotease secreted by E. faecalis, hydrolyzes gelatin, collagen, and casein, and has been implicated as a virulence factor in animal models.The ability of this enzyme to hydrolyze collagens and certain bioactive peptides suggests its participation in the initiation and propagation of inflammatory processes involving E. faecalis (Furumura et al., 2006).Gelatinase production was detected on nutrient gelatin, liquefaction of gelatin after placed nutrient gelatin in refrigerator considered a positive result.
The results of this study showed that 10% of E. faecalis isolates were gelatinase producer (Figure 1).This result was in agreement to the result of Al-Jmor (2012) that represented the percentage of gelatinase producer was 11.76%, whereas Vergis et al. (2002) showed that 64% of E. faecalis isolated from patients with bacteremia, produced gelatinase, while Jankoska et al. (2008) was reported that 68% of E. faecalis isolates were gelatinase producer.
Aggregation substance (AS) of E. faecalis is a pheromone inducible surface protein encoded by pheromone plasmids that facilitates conjugative exchange, by mediating strong binding between cells and formation of cell aggregates (clumps).AS also contributes to pathogenicity by enhancing  cell adhesion and internalization as well as favoring intracellular survival within macrophages (Glimore, 2002).Aggregation substance was detected on trypticase soy broth, the presence of adherent dense cell growth in the bottom of the tube was considered a positive result (Figure 2).
The results showed that all tested isolates (100%) have produced a slime layer by this method but in different levels of adherent layer which ranged from weak to moderate and strong (Figure 3).The result of this study was similar with the result reported by Al-Jmor (2012), the result of this study was higher than the result reported by Necidova et al., (2009)  A positive correlation between the presence of Esp and the ability of an Enterococcal strain to form biofilms in vitro has been reported.None of the esp-deficient isolates tested in that study were capable of forming biofilms.However, it was also observed that insertional inactivation of esp did not cause a loss of the biofilm phenotype in every mutant tested (Toledo-Arana et al., 2001).
The involvement of Enterococcal surface protein in biofilm formation in the presence of a higher glucose concentration has been reported by Tendolkar et al., (2004).Two E. faecalis esppositive strains and produce significantly more bio volume and thickness of biofilm than their controls, esp-negative strains.In the same study, the presence of 0.5% glucose in the growth medium influenced the biofilm production by E. faecalis (Tendolkar et al., 2006).
Di Rosa et al., (2006) have explained that neither esp seemed to be required for biofilm formation: both E. faecalis and did not show a correlation between the presence of esp and biofilm formation.The presence of esp and biofilm together was only found in strains from clinical settings, suggesting that there exists a synergy between this factor which serves as an advantage for the process of infection.

DNA and plasmid Extraction from E. faecalis isolates
The concentration results of DNA were ranged between 100-1800 ng/ml, while the purity of Genomic DNA was ranged between 1.8-1.9 in wavelength 260/280.This indicates the method was efficient in extraction and purification of DNA as shown in figure 4. The purpose of DNA extraction is for detection the virulence factor Enterococcal surface protein esp gene.

esp gene amplification by monoplex PCR technique
To detect the virulence factor Enterococcal surface protein esp gene in E. faecalis, Twenty E. faecalis isolates was subjected to PCR technique in a monoplex pattern.
The Positive result of esp gene was confirmed by 1.5% agarose gel electrophoresis stained with ethidium bromide, electrophoresed in 70 volt for 1.5 hrs and photographed under ultraviolet (UV) trans illuminator (figure [3][4][5][6][7][8][9][10]. The results of present study showed that esp gene band detected at 933 bp region, all E. faecalis isolates have esp gene as virulence factor. Figure (6)  In this study all E. faecalis isolates which is esp producer have produced biofilm which was ranged between strong to moderate and weak.This explains a strong positive correlation between the presence of esp and biofilm formation, and E. faecalis biofilm productivity dependent on esp expression.
Chuang-Smith et al. (2010) speculated that Esp may mediate the interaction with primary surfaces and participate in the formation of this phenotype.In addition, Heinkens et al. (2007) showed that Esp is involved in biofilm formation.
There are diverging opinions concerning the role of Esp in biofilm production (Garth et al. 2008).Dworniczek et al. (2012) concluded that there is no clear relationship between the expression of esp or gelE and biofilm forma tion.Indeed, an analysis by Sillanpaa et al. (2010) showed efficient biofilm production in the absence of Esp.
Comerlato et al. (2013) was showed there is no association between the presence of esp and biofilm production, they were assumed that other factors are associated with this phenotype.

PCR Technique
The accurate and rapid diagnosis of antibiotic resistance genes in the treatment of E. faecalis infections is extremely important in preventing the spread of infections.PCRbased molecular methods are often preferred for determination of antibiotics resistant genes.Using PCR technique, the genetic determinants of vancomycin resistant vanA and vanB were amplified to identify susceptible (lacking vanA or vanB) and resistant (vanA or vanB) for 13 E. faecalis isolates.
The results of the present study showed that, vanA gene bands were detected at 550 bp regions, Eleven E. faecalis isolates (84.6%) were produced 550 bp band (Figure 7), Four E. faecalis isolates (30.7%) produced vanB gene bands were nearly detected at 600 bp region, (Figure 8).
Regarding vanA gene bands, the sensitive isolate (1w.i) did not have vanA gene, since it is specific marker for vancomycin resistance.
vanA resistance is usually inducible by sub inhibitory concentrations of vancomycin and teicoplanin and it associated with production of a 38-40 kD membrane protein called VanA and encoded by vanA gene (Nicas et al., 1989).
The deduced amino acid sequence of vanA indicates that it is related to the bacterial D-Ala:D-Ala ligases, which mediate the D-alanyl-D-alanine (D-Ala-D-Ala) linkages of the growing cell wall peptidoglycan (Malen et al., 1990).VanA appears to be a D-Ala:D-X ligase of relaxed substrate specificity that can condense D-alanine with other amino acids, fatty acids, or hydroxy acids (Arthur et al., 1992).In resistant isolates, D-Ala-D-Ala is replaced with D-Ala-D-lactate (D-Lac), which cannot bind glycopeptides, The production of D-Ala-D-Lac depends on the cooperative activity of three enzymes, VanA, VanH, and VanX (Arthur et al., 1991).In vancomycin-resistant E. faecalis, the genes encoding these proteins are located on a plasmid and arranged sequentially within an operon.Induction of resistance is mediated by the transcription activator vanR and the membrane sensor vanS, located upstream of the other members of the gene cluster (Arthur et al., 1992;French, 1998).
The The results vanA in this study revealed that the gene have a genetic variation as showed in their molecular weight during electrophoresis of vancomycin resistant E. faecalis strains.
The vanB operon contains genes encoding a dehydrogenase, a ligase, and a dipeptidase, all of which have a high level of sequence identity (67%-76% identity) with the corresponding deduced proteins of the vanA operon, the function of the additional vanW protein found only in the vanB cluster is unknown (Patel et al., 1998;Dahl et al., 1999) The regulatory system in class B strains appears insensitive to induction by teicoplanin.Teicoplanin induces the synthesis of VanA-related proteins but does not induce the production of VanBrelated proteins.On the other hand, vancomycin induces the synthesis of the resistance proteins of both systems, and in fact, if a teicoplaninsusceptible Enterococcus with the vanB gene cluster is preexposed to vancomycin, the strain then tests teicoplanin resistant as well (Cetinkaya et al., 2000).
Regarding to genetic variation, the results of this study showed that vanB gene bands have molecular weight closely to 600 bp, while the results reported by Satak et al. vanA and vanB genes carried on Tn-1546 transposon that it is ubiquity, this gene could move (transpose) from one plasmid to other in the same or different bacterial strain, this explained by the conjugation experiment.The complete genome sequence of E. faecalis, a vancomycin-resistant clinical isolate, revealed that more than a quarter of the genome consists of probable mobile or foreign DNA.One of the predicted mobile elements is a previously unknown vanB vancomycin-resistance conjugative transposon (Paulsen et al., 2003).
In addition to transcriptional and translational regulatory mechanisms, many bacteria have evolved additional mechanisms to enable them to respond to changing environments.In these bacteria a genetically divers population is generated by reversible genetic changes known as phase variation, therefore; these isolates to survive in their environment, it must gain a new characters that enable them for the survivor, they are gained these characters with missing some of unimportant sequences from genes that still activated in absence these sequences (Dale and park, 2003).

Detection of vanA, vanB genes using Multiplex PCR Technique
The multiplex PCR assay in our hands is a convenient and rapid method for determining glycopeptide resistance genotypes for Enterococcus spp. in the clinical microbiology laboratory.The assay provides a more specific and rapid alternative to classical phenotypic methods for the detection of low level glycopeptide resistance (MIC range, 4 to 8 mg/ml) (Patel et al. 1997).
Figure 3-13 demonstrated that Twelve (95%) E. faecalis isolates out of thirteen isolates were obtained from clinical samples found to be vancomycin resistant isolates, Eleven E. facalis isolates (84.6%) have vanA gene bands were detected at 550bp regions, Four E. faecalis isolates (30.7%) have vanB gene bands were detected nearly at 600bp region.Three of E. faecalis isolates (5U, 7U and 3R) were having vanA-vanB cluster.
All E. faecalis isolates were showing vanA or vanB genotype in PCR assay, no other genotype were showed because of only the extracted pHT² plasmids were amplified in PCR for detection vanA or vanB genes or both, the other van genes are chromosomally.
The results of MIC showed a perfect similarity with the results of PCR, this reflect the efficiency of MIC method.Vancomycin-sensitive E. faecalis isolates were have MIC of vancomycin 4 µg/ml and they were havn't any van genes in gel electrophoresis of multiplex PCR product, While the Intermediate-resistant E. faecalis isolates showed vanA genotype and its MIC was 16 µg/ml (Table 3-6).The resistant E. faecalis isolates have shown identical results between MICs and PCR which was varied in its van genes, these isolates showed vanA, vanB or vanAvanB cluster genotypes.While it's MIC was e"32.
R e s i s t a n c e t o g l y c o p e p t i d e s i s disseminating rapidly and has recently spread to methicillin-resistant S. aureus (Sievert et al. 2002).Rapid and accurate methods are thus essential for the detection of such clinical isolates and the prevention of their transmission.In addition, MIC determination is time-consuming and does not detect GRE with low-level glycopeptide resistance and misidentification of E. faecalis can occur with commercial systems (Willey et al, 1999;Depardieu et al. 2004).
One of the limitations of the method proposed, or of other similar assays, could be the sequence variability among van genes that has occasionally been observed (Perichon et al. 1997).

Fig. 6 .
Fig. 6.Gel electrophoresis of amplified PCR product of vanA gene (550 bp) of E. faecalis isolates in monoplex pattern

Fig. 8 .
Fig. 8. Gel electrophoresis of amplified PCR product of vanA, vanB genes of E. faecalis isolates in multiplex pattern Figure3-12shows that the molecular weight of vanB gene bands were near 600 bp region, Vancomycin resistant E. facalis strains and their MIC were (5U and 3R=64 µg/ml, 4R=128 µg/ml and 7U=32 µg/ml).The vanB operon contains genes encoding a dehydrogenase, a ligase, and a dipeptidase, all of which have a high level of sequence identity (67%-76% identity) with the corresponding deduced proteins of the vanA operon, the function of the additional vanW protein found only in the vanB cluster is unknown(Patel et al., 1998;Dahl et al., 1999) The regulatory system in class B strains appears insensitive to induction by teicoplanin.Teicoplanin induces the synthesis of VanA-related proteins but does not induce the production of VanBrelated proteins.On the other hand, vancomycin induces the synthesis of the resistance proteins of both systems, and in fact, if a teicoplaninsusceptible Enterococcus with the vanB gene cluster is preexposed to vancomycin, the strain then tests teicoplanin resistant as well(Cetinkaya et al., 2000).Regarding to genetic variation, the results of this study showed that vanB gene bands have molecular weight closely to 600 bp, while the results reported by Satak et al. (1997); Depardieu et al. (2004); Biendo et al. (2010); Tellis and Muralidharan, (2012), they were detected vanB gene in E. faecalis isolates and the molecular weight was 635 bp.While Clark et al. (1993); Kariyama et al. (2000) were reported the molecular weight of vanA gene from PCR product was 433 bp.Miele et al. (1995); Patel et al. (1997) were reported that molecular weight of vanA gene from PCR product was 457bp, 885 bp, respectively.vanAand vanB genes carried on Tn-1546 transposon that it is ubiquity, this gene could move (transpose) from one plasmid to other in the same or different bacterial strain, this explained by the conjugation experiment.The complete genome sequence of E. faecalis, a vancomycin-resistant clinical isolate, revealed that more than a quarter of the genome consists of probable mobile or foreign DNA.One of the predicted mobile elements is a previously unknown vanB vancomycin-resistance conjugative transposon(Paulsen et al., 2003).In addition to transcriptional and translational regulatory mechanisms, many bacteria

Table 1 .
Number and Nomenclature of bacterial isolates

Table 2 .
The primers and their sequences used in conventional PCR

Table 4 .
Program was processed in PCR amplification of vanB gene according to Dutka-Malen et al., 1995

Table 6 .
Program was processed in Multiplex PCR amplification of vanA and vanB genes

Table 7 .
Numbers and percentages of E. faecalis isolates from clinical specimens

Table 8 .
The Minimum Inhibitory Concentrations [MICs] of Vancomycin for E. faecalis isolates S: Sensitive, IR: Intermediate Resistant, R: Resistant.

Table 9 .
Multi-drug resistant pattern of E. faecalis isolates and Oli et al., (2012) who found that 28% and 85% of E. faecalis isolates were produce slime layer, respectively.The results in figure (1) , showed that all E. faecalis isolates (100%) were AS producer.The result of this study was similar to Paoletti et al. (2007) was showed that 100% of E. faecalis isolates were AS producer, While Tomita and Ike (2008) were reported (33%) of E. faecalis isolates have the ability to produce AS.Alebouyeh et al. (2005) was showed 23.3% of E. faecalis isolates were AS producer.
demonstrated that the twenty (100%) of E. faecalis isolates were obtained from clinical samples found it has esp gene, Shankar et al. (1999) was reported that 26.3% of E. faecalis isolates having the virulence factor ESP. Creti et al. (2004) and Jankoska et al. (2008) were reported that E. faecalis which have the esp gene was represented to 44.6% and 76%, respectively.